Modulation of growth hormone receptor expression and insulin-like growth factor expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of growth hormone receptor and/or insulin like growth factor-I (IGF-I). The compositions comprise oligonucleotides, targeted to nucleic acid encoding growth hormone receptor. Methods of using these compounds for modulation of growth hormone receptor expression and for diagnosis and treatment of disease associated with expression of growth hormone receptor and/or insulin-like growth factor-I are provided. Diagnostic methods and kits are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/451,455, filed Feb. 28, 2003, and U.S. Provisional Application No.60/490,230, filed Jul. 24, 2003, the entire disclosures of which arehereby incorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of growth hormone receptor. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodinggrowth hormone receptor. Such compounds are shown herein to modulate theexpression of growth hormone receptor and also to modulate theexpression of insulin-like growth factor 1 (IGF-I) to animal and humanequivalent therapeutic levels which are relevant to the treatment ofdiseases including acromegaly, gigantism, age-related maculardegeneration, diabetic retinopathy, diabetic nephropathy, diabetes, andgrowth hormone and IGF-I dependent tumors. The growth hormone receptormodulating effects are also relevant to the treatment of arthritis andother conditions involving growth hormone receptor and/or growthhormone/insulin-like growth factor-I axis. Similarly, antisensecompounds directed to any one or more of the targets in the growthhormone/insulin-like growth factor-I axis, including growth hormone,growth hormone receptor, IGF-I and IGF-I receptor, can be used in thetreatment of the same conditions.

BACKGROUND OF THE INVENTION

Growth hormone, released by the pituitary, is a member of a cascade ofhormones that regulate growth of the body and its organs. Secretion ofgrowth hormone into the bloodstream is followed by binding to growthhormone receptor (GHR) on many cell and organ types. Growth hormonesignaling is mediated by this interaction. Growth hormone signalingcauses the production of another hormone, insulin-like growth factor-I(IGF-I or IGF-1), which is produced in the liver, adipose tissue andkidney and secreted into the bloodstream. About 75% of serum IGF-I isproduced in the liver in response to growth hormone stimulation. Manydisorders are caused by and/or associated with elevated growth hormonelevels and/or elevated IGF-I levels in plasma and/or tissues includingacromegaly, gigantism, retinopathy, macular degeneration, nephropathy,diabetes and cancers. This role of IGF-I in mediating many growthhormone effects is well recognized and the interrelationship is referredto as the growth hormone/insulin-like growth factor-I axis. In a normalfeedback loop, IGF-I also causes the production of growth hormone by thepituitary to be reduced.

Growth hormone is produced and secreted by a set of specialized cells inthe anterior pituitary. Growth hormone has direct and indirect effectson many tissues, such as stimulating bone and soft tissue growth andinfluencing carbohydrate, protein, and lipid metabolism. Directbiological activities of growth hormone include receptor binding,internalization of the hormone/receptor complex, and activation ofproteins involved in signal transduction.

Protein and RNA transcripts for receptors of growth hormone (GHR) havebeen detected in many of the tissues influenced by the hormone. It wasdetermined that a single molecule of growth hormone binds sequentiallyto two receptor molecules, forming an active′ complex. This complex, inturn, signals stimulation of other genes, including IGF-I. IGF-I,produced and secreted by the liver and other target tissues, mediatessome of the indirect effects of growth hormone on growth anddevelopment. Other intracellular events occurring after the growthhormone/growth hormone receptor interaction include activation oftyrosine kinases such as Janus kinase 2 (Jak-2), which leads tophosphorylation and activation of other proteins including signaltransducer and activator of transcription 5A and 5B (STAT 5A and 5B) andmitogen activated protein (MAP) kinase that, in turn, activate otherproteins and genes.

The cDNA encoding the growth hormone receptor has been cloned from manyspecies. The receptor consists of an extracellular hormone-bindingregion (exons 2-7), a single membrane spanning region (exon 8), and anintracellular region (exons 9-10). There are also multiple alternative5′ untranslated regions which are alternative first exons of the gene,in both the human and mouse transcripts. Growth hormone receptor has nointrinsic kinase domain, but the intracellular region plays a major rolein the signal transduction process. A truncated form of the receptor,known as growth hormone binding protein (GHBP), lacks the transmembraneand intracellular regions of growth hormone receptor and is secretedinto the serum. The truncated protein is produced by one of twodifferent processes, depending on the animal species. In mice and rats,alternative splicing of growth hormone receptor precursor messenger RNAreplaces the transmembrane and intracellular regions with a very shorthydrophilic tail (encoded by exon 8A; 15, 16). In humans, cows, and pigs(among others), no alternative RNA splicing is apparent but instead theGHBP is produced by proteolysis of the growth hormone receptor. Thefunction of the binding protein appears to be to modulate the level ofcirculating growth hormone.

Growth hormone receptor is expressed in many organs and tissuesincluding liver, adipose tissue, muscle, cartilage, bone, tooth, kidney,eye, cardiovascular system, gut, reproductive organs, skin, brain,endocrine system and immune system.

The three-dimensional structure of the extracellular domain of growthhormone receptor has been established. It consists of two modules, eachof about 100 amino acids, arranged as two sandwiches each with sevenstrands of beta-sheet. The secreted form of the extracellular domain ofgrowth hormone receptor is the GHBP.

The growth hormone receptor is biologically responsive to growth hormonestimulation. JAK2 is the primary effector molecule for growth hormonereceptor signaling. JAK2 is activated post growth hormone receptordimerisation. When the growth hormone dimerizes its receptors, the JAKsare brought close together, and with proper alignment transphosporylateeach other, leading to full activation. The intracellular targets forthe JAKs include tyrosine residues in the receptor cytoplasmic domainitself, which in turn activate SH2 domains (STATS, Shc and SHP2). Thesemay go on to activate the MAP kinase pathway, which regulates cellproliferation. JAK2 also phosphorylates and activates other signalingmolecules, such as IRS-1 and -2 and phosphatidyl 3-inositol kinase,which are important parts of the insulin signaling mechanism and mayaccount for the insulin-like actions of growth hormone. Activated JAK2also phosphorylates STAT5, and when activated, is involved in thetranscription of a number of genes.

Growth hormone receptor activation leads to many actions in many organsincluding the following outcomes in the following organs:

Liver: Increased secretion of insulin-like growth factor-I, synthesis ofplasma proteins, regulation of nitrogen balance enzymes, increasedcarbohydrate synthesis/storage, and increased fat breakdown; AdiposeTissue: Breakdown of fat stores; Muscle: Increased protein synthesis,decreased protein breakdown; Cartilage: Increased height by increasingproliferation and differentiation of chondrocytes in growth plate; Bone& Tooth: Increased turnover of tissue, both synthesis and breakdown;Kidney: Increased sodium, bicarbonate and water retention; Eye:increased retinal neovascularization; Cardiovascular: Hypertrophy,increased contractility, stroke volume, cardiac output; Gut:Hypertrophy, increased amino acid, sodium, calcium, phosphate and B12uptake; Reproductive System: Increased sperm production and motility,increased accessory gland secretion in male, increased number offollicles and ovulation rate, increased follicular maturation rate,increased milk production; Skin: Increased skin thickness and strength,increased hair growth and thickness; Brain: Increased neuronproliferation and connectivity prenatally, increased myelin formation,improved long-term memory; Endocrine System: Increased insulin synthesisand secretion, increased adrenal steroidogenesis; Immune System:Increased immune cell proliferation, increased killing by monocytes,macrophages and NK cells, increased antibody production.

Downstream from growth hormone receptor in the growth hormone signalingpathway are IGF-I and IGF-I receptor. The insulin-like growth factors(IGFs) are important in proliferation. In particular, IGF-I and IGF-2are ubiquitous polypeptides each with potent mitogenic effects on abroad range of cells. Molecules of the insulin-like growth factor typeare also known as “progression factors” promoting “competent” cellsthrough DNA synthesis. The insulin-like growth factors act through acommon receptor known as the Type I receptor or IGF-IR, which istyrosine kinase linked.

Particular proteins, referred to as insulin-like growth factor bindingproteins (IGFBPs), appear to be involved in autocrine/paracrineregulation of tissue insulin-like growth factor availability (Rechlerand Brown, Growth Regulation, 1992, 2, 55-68). Six IGFBPs have so farbeen identified. The exact effects of the IGFBPs are not clear andobserved effects in vitro have been inhibitory or stimulatory dependingon the experimental method employed (Clemmons, Growth Regn. 1992, 2,80,). There is some evidence, however, that certain IGFBPs are involvedin targeting insulin-like growth factor-I to its cell surface receptor.Also expression of IGFBP-3 is regulated by growth hormone (Karen et al,supra).

The IGF-IR is a tyrosine kinase linked cell surface receptor (Ullrich etal., EMBO J. 1986, 5, 2503-2512,) that regulates cell division,transformation and apoptosis in many cell types (LeRoith et al., Endocr.Rev., 1995, 16, 143-163; Rubin and Baserga, Laboratory Investigation,1995, 73, 311-331).

If feedback regulation of growth hormone production is lost and thepituitary continues to release aberrant amounts of growth hormone, thelevel of insulin-like growth factor-I continues to rise, leading to bonegrowth and organ enlargement. The excess growth hormone also causeschanges in sugar and lipid metabolism, which may lead to diabetes.Defects in the growth hormone signalling pathway often lead toabnormalities of stature and body and/or organ size. Mutations in thegrowth hormone receptor gene result in extreme short stature (Laron'ssyndrome). Excessive production of growth hormone can lead to acromegalyor gigantism.

Acromegaly and gigantism are related growth disorders wherein growthhormone excess, sometimes caused by pituitary tumor, causes progressivecosmetic disfigurement and systemic organ manifestations. It affects40-50 per million people worldwide with about 15,000 sufferers in eachof the UE and Europe and an annual incidence of about 4-5 per million.It is initially characterized by abnormal growth of the hands and feetand bony changes in the facial features. Patients have reduced qualityof life with overgrowth of the jaw, enlargement of hands and feet,deepening of the voice, thickening of skin, offensive body odor,articular cartilage problems, hyperphosphatemia, peripheralneuropathies, higher blood pressure, diabetes, heart disease, andcancer, and have a reduced life expectancy if untreated. The mortalityrate is about twice that of the normal population due tocardiorespiratory and cardiovascular diseases, diabetes and neoplasia,particularly colon cancer. The goal of current treatment is to reversethe effects of the hypersecretion of growth hormone and normalizeproduction of IGF-I which is elevated by about 50% in these patients.When effective, treatment moderates disease symptoms anddisease-associated mortality.

Gigantism, the disease of excess growth hormone in children, is a raredisorder. In gigantism, excessive linear growth occurs whilst epiphysealgrowth plates are open during childhood with growth hormone excesscaused via a benign pituitary tumor. In both gigantism and acromegaly,all growth parameters are affected, although not necessarilysymmetrically. Many of the growth related outcomes are mediated byelevated levels of serum IGF-I. Serum blood levels of IGF-I are elevatedby about 50% in patients and reduction of serum IGF-I is used to monitortreatment success.

Treatments for acromegaly and gigantism involve the ability to lower theelevated IGF-I in plasma. This may be achieved by surgical removal andradiation therapy of the benign pituitary tumor but this is effective inonly 50% of patients. Dopamine agonists such as bromocriptine mesylateor cabergoline may be dosed orally which is convenient but they onlyreduce growth hormone production and associated IGF-I sufficiently in10% of cases. They also produce significant gastrointestinal and centralside effects in 20-30% of patients. Also used in treatment of acromegalyare the somatostatin analogues such as Sandostatin or octreotide, whichinhibit the release of growth hormone releasing hormone (GHRH) from thehypothalamus, and/or pituitary and thereby reducing production of growthhormone in the pituitary. This compound is effective in 60-65% patientswith acromegaly but it must be injected under the skin every 8 hours orintramuscularly for effective treatment.

Recently a growth hormone receptor antagonist, Trovert, also known asSomavert, Pegvisomant and B2036-PEG, was shown in clinical trials to beeffective in 90-95% of patients. Clinical trial experience to date showsa 10% drop-out rate and adverse effects such as liver dysfunction.Trovert is a growth hormone molecule with a 9 amino acid substitutionwith 4-5 pegylations to increase half life. Like all modified proteinsit is immunogenic, with antibodies being made to Trovert within 1 monthof dosing. This can impact Trovert's short and long term utility andmakes dosing difficult to predict. Trovert was initially dosed once permonth by subcutaneous (sc) administration, but current clinical practicesuggests dosing will need to be once/day sc. Trovert interferes withgrowth hormone binding to its receptor but not the Growth HormoneBinding Protein (GHBP) fragment of the growth hormone receptor. GHBPbinds growth hormone prolonging its action, which can be disadvantageousin conditions involving excess growth hormone and/or excess IGF-I.Pegylation may also impact on Trovert's long term safety profile.

Diabetes and its life threatening complications such as diabeticretinopathy and nephropathy are also disorders associated with growthhormone and/or IGF-I levels. First line treatment of these conditionsinvolves controlling hyperglycemia. Drugs that control diabetes reducethe incidence of nephropathy by 60% and also reduce the incidence ofretinopathy. However, about half of all diabetics are unaware of diseaseand therefore remain untreated, so diabetic nephropathy and retinopathyare likely to remain a major condition requiring other treatments. Inretinopathy surgical ablative treatments such as laser pan-retinalphotocoagulation are used but these remain incompletely effective anddestroy retinal tissue, causing partial vision field loss. In type Idiabetics ACE and AII inhibitors decrease albuminuria excretion byacting on the kidney and in Type II diabetics the same inhibitors actlocally on kidney and also decrease blood pressure to reduce the risk ofdeath from kidney failure by another 50%. However, 20-30% of patientsremain resistant to treatment with current glycemic control drugs andACE drugs. There is thus a need for better treatments.

The underlying cause of diabetes, diabetic retinopathy and diabeticnephropathy may be insulin related hyperglycemia, but growth hormoneand/or insulin-like growth factor-I excess is also important. Octreotideinhibitors of GHRH that decrease production of pituitary growth hormone,reducing systemic levels of growth hormone and IGF-I, and/or modulatinglocal tissue levels show potential in the clinic. A study withoctreotide by Grant et al., Diabetes Care, 2000, 2, 504-9) reducingsIGF-1 by 51% at maximally tolerated doses of octreotide 5000 μg/day screduced the need for laser surgery in retinopathy patients to 1 patientout of 22 rather than 9/22 in placebo in a 15 month study. Also oculardisease was reduced to 27% vs placebo of 42% bordering on significance(P 0.06). Three human studies using octreotide at levels that reducedsIGF1 45%, about 20% and about 10% respectively were at least partlyeffective in clinical trials of nephropathy. The outcome reported bySerin et al. (JAMA, 1991, 265, 888-92) with 11 patients used high dosesof octreotide in a 12 week study that reduced serum IGF-I by 45%. At thetime it was stated to be the best effect observed on reducing glomerularfiltration rate with a 22-33% reduction relative to placebo. This dose,however, was near maximally tolerated doses of octreotide.

Animal pathology model studies with octreotide and Trovert also supportthe view that agents that modulate the growth hormone/insulin-likegrowth factor-I axis are beneficial in the treatment of these diabeticconditions. Growth hormone and its receptor are implicated in theinduction of glomerular hypertrophy and sclerosis in partial nephrectomyand diabetic nephropathy with somatostatin inhibitors octreotide andPTR-3173 (Groenbaek et al., J. Endocrinol., 2002, 172, 637-643 andLandau et al, Kidney International, 2001, 60, 505-512) and growthhormone receptor antagonist, G120K-PEG, a weaker version of Trovert,preventing complications in type I and Type II diabetic mice (Chen etal, Endocrinology, 1996, 137, 11, 5136-5165; Flyvbjerg et al, Diabetes,1999, 40,377-382, and Segev et al., J. Am. Soc. Nephrol. 1999,10,2374-81). Growth hormone and its receptor are implicated in theinduction of retinal neovascularization through IGF-I with somatostatininhibitors octreotide and growth hormone receptor antagonist MK678,inhibiting retinal neovascularization in mice. MK678 reduction ofneovascularization correlated with low serum IGF-I (Smith et al,Science, 1997, 276, 1706-9). Oxygen induced retinopathy in the mouse wasalso responsive to octreotide as reported by Higgins et al., Exp. EyeRes, 2002, 74,553-9.

Macular degeneration is also associated with elevated growth hormoneand/or IGF-I levels. Age-related macular degeneration (AMD) is caused bydeterioration of the central part of the retina, the macula, resultingin loss of detailed vision. Wet AND, the less common form, is caused byleakage from new blood vessels growing behind the retina. The growthhormone/IGF-I axis is involved in formation of new blood vesselsrelevant to this condition and to diabetic retinopathy.

Various cancers are also associated with aberrant growth hormone and/orIGF-I levels. Reduction of serum IGF-I by 20-50% using Trovert decreasedtumor volume in breast cancer in animal models and helped in coloncancer, liver metastasis, and meningiomas (Friend et al, Proceedings11th NCI EORTC. AACR Symposium and Friend, Growth norm. IGF Res., 2001,Jun. 11 Suppl A: S121-3). The incidence of breast, colon, prostate, andlung cancer is increased in individuals in the high normal range ofserum IGF-I. There have been no clinical studies with Trovert incancers. However, octreotide is indicated for gastro-pancreatic cancers.

Other conditions that may be associated with elevated growth hormoneand/or IGF-I levels include rheumatoid arthritis. A pilot clinical studyshowed octreotide was useful for the treatment of active refractoryrheumatoid arthritis in a subset of patients (Paran et al., Ann. Rheum.Dis., 2001, 60, 888-91. with comments and authors' reply in Ann. Rheum.Eds., 2002, 61, 1117).

Longevity may also be improved with modulation of growth hormonereceptor (Coschigano et al., Endocrinology, 2000, 141, 2608-2613). Therewas a significant increase in lifespan of nearly a year in doubleknockout animals with low levels of IGF-I and high levels of growthhormone.

Another application to modifying levels of growth hormone and/or IGF-Ivia the growth hormone receptor may enable stem cell differentiationtowards neural cell production as growth hormone inhibits neuronaldifferentiation of neural progenitor cells (Turnley et al., NatureNeuroscience, 7 Oct. 2002, published online). Other applications will beknown to those skilled in the art.

Although the underlying roles in various disease or conditions may bedifferent, the above conditions arise at least in part from incorrectlevels of expression of local and/or systemic growth factors growthhormone and IGF-I and/or their receptors growth hormone receptor andIGF-IR. In these situations, dopamine agonists, somatostatinantagonists, and growth hormone receptor antagonists targeting theproteins have been used and/or shown potential.

While a range of treatments have been developed for agents that modifythe growth hormone-insulin-like growth factor axis, and growth hormonereceptor and IGF-IR, none is completely effective and/or free of adverseside effects. Moreover, there is potential disadvantages in the routesand/or frequencies of administration that can affect compliance.

It is therefore an object of the present invention to provide novelproducts and compositions wherein one or more of the above problems andlimitations are ameliorated.

In the last decade, there have been reports of the use of antisenseoligonucleotides to explore gene function and several reports in thedevelopment of nucleic acid based drugs. Antisense oligonucleotidesinhibit mRNA translation via a number of alternative ways includingdestruction of the target mRNA through RNase H recruitment, orinterference with RNA processing, nuclear export, folding or ribosomescanning.

Pellegrini et al. attempted to block growth hormone receptor synthesisin the central nervous system by infusing intracerebroventricularly anantisense 18-mer oligonucleotide complementary to a portion of thecoding sequence of the rat growth hormone receptor mRNA overlapping thetranslation initiation codon. J. Neurosci. 1996, 16, 8140-8148.

The current invention as exemplified herein for the first time,demonstrates that an antisense oligonucleotide targeted specifically tothe growth hormone receptor reduces a clinical parameter of growthhormone activity, namely serum insulin-like growth factor-I.Importantly, our antisense studies teach the ability to use antisense togrowth hormone receptor to reduce serum insulin-like growth factor-I bysimilar degrees required for the clinical treatment of gigantism oracromegaly. Serum insulin-like growth factor-I levels are elevated inacromegaly patients and reduced at human therapeutic Trovert doses by50% in both 12 week studies (Trainer et al, The New England J of MedApr. 20, 2000) which show a decrease by 1.3 to 2 fold, and in long termgreater than 1 year studies as reported by van der Lely et al., Lancet2001, Nov. 24: 358 (9295) 1754-1759.

Similar levels of reduction of serum insulin-like growth factor-I arealso reported with octreotide in 15 month clinical trials of diabeticretinopathy (Grant et al, Supra) and in clinical trials in diabeticnephropathy (Serri et al, supra). Similar levels of reduction of 20-50%is also sufficient to prevent the growth of certain cancer in animalmodels (Friend, supra).

The present invention teaches for the first time that growth hormonereceptor antisense can achieve human and animal equivalent therapeuticoutcomes. It teaches that antisense to the mRNA of one component of thegrowth hormone/insulin-like growth factor-I axis, namely growth hormonereceptor, can affect another parameter in the axis, e.g., IGF-I.Importantly, it teaches that antisense targeting any other target in,the growth hormone/insulin-like growth factor-I axis is potentiallycapable of achieving therapeutic levels in conditions dependent onexcess growth hormone or insulin-like growth factor-I levels.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding growth hormone receptor, and which modulate growth hormonesignaling or the growth hormone/insulin-like growth factor-I axis,particularly the expression of growth hormone receptor and/orinsulin-like growth factor-I. Further provided are methods of screeningfor modulators of growth hormone receptor and/or insulin-like growthfactor-I and methods of modulating the expression of growth hormonereceptor and/or insulin-like growth factor-I in cells, tissues oranimals comprising contacting said cells, tissues or animals with one ormore of the compounds or compositions of the invention. Diagnosticmethods and kits are also provided. Methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with growth hormone signaling or the growthhormone/insulin-like growth factor-I axis, particularly the expressionof growth hormone receptor and/or insulin-like growth factor-I, are alsoset forth herein.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding growth hormone receptor. This is accomplished byproviding oligonucleotides which specifically hybridize with one or morenucleic acid molecules encoding growth hormone receptor. As used herein,the terms “target nucleic acid” and “nucleic acid molecule encodinggrowth hormone receptor” have been used for convenience to encompass DNAencoding growth hormone receptor, RNA (including pre-mRNA and mRNA orportions thereof (including both coding and noncoding regions),transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofgrowth hormone receptor. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition isoften the preferred form of modulation of expression and mRNA is often apreferred target nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70% sequence complementarity to atarget region within the target nucleic acid, more preferably that theycomprise 90% sequence complementarity and even more preferably comprise95% sequence complementarity to the target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid. One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown thatthe primary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507).The posttranscriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 2002, 295, 694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes growth hormone receptor.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding growth hormone receptor, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

In mouse, rat and monkey, growth hormone binding protein, which is thesoluble shortened form of growth hormone receptor, is produced byalternative splicing of the growth hormone receptor primary transcript.In some embodiments it may be preferred to target regions of thetranscript which are present in both the growth hormone receptortranscript and in the shorter growth hormone binding protein transcript.In other embodiments it may be preferable to target regions of the mRNAwhich are only present in the longer growth hormone receptor transcript.In humans, cows, and pigs (among others), no alternative RNA splicing isapparent but instead the shorter growth hormone binding protein isproduced by proteolysis of the growth hormone receptor. It will beunderstood that in the context of this invention, “nucleic acid encodinggrowth hormone receptor” thus includes nucleic acid encoding growthhormone binding protein.”

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The growth hormone receptor mRNA has alternative 5′ untranslated regionsand one or more of these may be preferred for targeting.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target region's, segments or sites have beenidentified, antisense compounds are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired effect.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of growth hormone receptor. “Modulators” arethose compounds that decrease or increase the expression of a nucleicacid molecule encoding growth hormone receptor and which comprise atleast an 8-nucleobase portion which is complementary to a preferredtarget segment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding growthhormone receptor with one or more candidate modulators, and selectingfor one or more candidate modulators which decrease or increase theexpression of a nucleic acid molecule encoding growth hormone receptor.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding growth hormone receptor, the modulatormay then be employed in further investigative studies of the function ofgrowth hormone receptor, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between growth hormone receptor and a disease state,phenotype, or condition. These methods include detecting or modulatinggrowth hormone receptor comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of growth hormone receptor and/or arelated phenotypic or chemical endpoint at some time after treatment,and optionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Cells, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A, 2000, 97, 1976-81), protein arrays and proteomics (Cells, et al.,FEES Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Cells, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding growthhormone receptor. For example, oligonucleotides that are shown tohybridize with such efficiency and under such conditions as disclosedherein as to be effective growth hormone receptor inhibitors will alsobe effective primers or probes under conditions favoring geneamplification or detection, respectively. These primers and probes areuseful in methods requiring the specific detection of nucleic acidmolecules encoding growth hormone receptor and in the amplification ofsaid nucleic acid molecules for detection or for use in further studiesof growth hormone receptor. Hybridization of the antisenseoligonucleotides, particularly the primers and probes, of the inventionwith a nucleic acid encoding growth hormone receptor can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of growth hormone receptor in a sample may also beprepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals.

The compounds of the present invention have been shown to reduceexpression of growth hormone receptor and to reduce levels of IGF-I.These compounds are therefore believed to be useful for prevention,delay or treatment of conditions associated with growth hormone receptoror with the growth hormone/insulin-like growth factor-I axis, includingacromegaly, gigantism, age-related macular degeneration, diabeticretinopathy, diabetic nephropathy, diabetes, arthritis and growthhormone and IGF-I dependent tumors.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofgrowth hormone receptor is treated by administering antisense compoundsin accordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of a growthhormone receptor inhibitor. The growth hormone receptor inhibitors ofthe present invention effectively inhibit the activity of the growthhormone receptor protein or inhibit the expression of the growth hormonereceptor protein. In one embodiment, the activity or expression ofgrowth hormone receptor in an animal is inhibited by about 10%.Preferably, the activity or expression of growth hormone receptor in ananimal is inhibited by about 30%. More preferably, the activity orexpression of growth hormone receptor in an animal is inhibited by 45%or more.

For example, the reduction of the expression of growth hormone receptormay be measured in serum, adipose tissue, liver or any other body fluid,tissue or organ of the animal. Preferably, the cells contained withinsaid fluids, tissues or organs being analyzed contain a nucleic acidmolecule encoding growth hormone receptor protein and/or the growthhormone receptor protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphoro-dithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may, be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleotides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents. Sodium is a suitable pharmaceutical salt,particularly for oligonucleotide compounds.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Also preferred antisense compounds are those capable of oraladministration such as the 2′MOE antisense compounds and morpholinophosphorodiamidates. This provides further convenience for usersrelative to growth hormone receptor compounds in the prior art.Preferred compounds in the treatment of some conditions will be thosethat distribute broadly and thus capable of both local and/or systemiceffects via the liver. It will be understood however, that in otherconditions distribution to fewer organs may be preferred.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998),Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filedFeb. 8, 2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine ara-binoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially. Particularly preferredcombinations comprise Octreotide, Trovert and/or other inhibitor(s) orantagonists of growth hormone, insulin-like growth factor-I, IGFBP-3,growth hormone receptor or insulin-like growth factor1 receptor.

Compositions of the invention may contain one or more antisensecompounds, particularly oligonucleotides, targeted to a first nucleicacid and one or more additional antisense compounds targeted to a secondnucleic acid target. Alternatively, compositions of the invention maycontain two or more antisense compounds targeted to different regions ofthe same nucleic acid target. Numerous examples of antisense compoundsare known in the art. Two or more combined compounds may be usedtogether or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

Preferred antisense oligonucleotides are made with chemistries capableof low frequency of dosing, i.e., once a day, once a week or less often.Particularly preferred antisense chemistries are those used herein whichmay be dosed once every second day and able to be dosed at least onceper week cc, if not less frequently at once per month, based on theobservations of antisense of the same class. This is less frequentlythan Trovert in same animal model, which was dosed every day, and lessfrequently than current clinical experience with Trovert. This providesenormous convenience for treatment of this chronic condition which maypotentially improve compliance.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyauridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-text-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleotide Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers' are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scared, 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric

Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(Methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingGrowth Hormone Receptor

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target growth hormone receptor. Inone embodiment these nucleic acid duplexes are double-stranded RNAcompounds (small interfering RNAs or siRNAs). In general, active sitesfor RNase H-dependent antisense oligonucleotides predict active sitesfor siRNA (Vickers et al., 2003, J. Biol Chem. 278, 7108-7118). In oneembodiment of the invention, the nucleobase sequence of the antisensestrand of the duplex comprises at least a portion of an oligonucleotidesequence shown in Table 1. Alternatively, a new “gene walk” in which aseries of dsRNAs targeted to growth hormone receptor are synthesized andtested may be used.

The ends of the dsRNA strands may be modified by the addition of one ormore natural or modified nucleobases to form an overhang. The sensestrand of the dsRNA is then designed and synthesized as the complementof the antisense strand and may also contain modifications or additionsto either terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini. The duplex may be aunimolecular or bimolecular duplex; i.e, the two strands may beconnected to each other directly or by means of a linker, or may beseparate molecules.

By way of example, a duplex comprising an antisense strand having thesequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:

  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG may be prepared with blunt ends(no single stranded overhang) as shown:

cgagaggcggacgggaccg Antisense Strand |||||||||||||||||||gctctccgcctgccctggc Complement

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate growth hormone receptor expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

MCF7:

The human breast carcinoma cell line MCF-7 was obtained from theAmerican Type Culture Collection (Manassas, Va.). MCF-7 cells wereroutinely cultured in DMEM low glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Growth HormoneReceptor Expression

Antisense modulation of growth hormone receptor expression can beassayed in a variety of ways known in the art. For example, growthhormone receptor mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA. Thepreferred method of RNA analysis of the present invention is the use oftotal cellular RNA as described in other examples herein. Methods of RNAisolation are well known in the art. Northern blot analysis is alsoroutine in the art. Real-time quantitative (PCR) can be convenientlyaccomplished using the commercially available ABI PRISM™ 7600, 7700, or7900 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Protein levels of growth hormone receptor can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), enzyme-linked immunosorbentassay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodiesdirected to growth hormone receptor can be identified and obtained froma variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalmonoclonal or polyclonal antibody generation methods well known in theart.

Reduction in expression of growth hormone receptor may also beindirectly measured by measuring decreases in insulin-like growthfactor-I in serum or other bodily fluid, tissues or organs.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof Growth Hormone Receptor Inhibitors Phenotypic Assays

Once growth hormone receptor inhibitors have been identified by themethods disclosed herein, the compounds are further investigated in oneor more phenotypic assays, each having measurable endpoints predictiveof efficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of growth hormone receptor in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with growthhormone receptor inhibitors identified from the in vitro studies as wellas control compounds at optimal concentrations which are determined bythe methods described above. At the end of the treatment period, treatedand untreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the growth hormone receptorinhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor growth hormone receptor inhibitor. Furthermore, to prevent thedoctors from being biased in treatments, they are not informed as towhether the medication they are administering is a growth hormonereceptor inhibitor or a placebo. Using this randomization approach, eachvolunteer has the same chance of being given either the new treatment orthe placebo.

Volunteers receive either the growth hormone receptor inhibitor orplacebo for eight week period with biological parameters associated withthe indicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodinggrowth hormone receptor or growth hormone receptor protein levels inbody fluids, tissues or organs compared to pre-treatment levels. Othermeasurements include, but are not limited to, indices of the diseasestate or condition being treated, body weight, blood pressure, serumtiters of pharmacologic indicators of disease or toxicity as well asADME (absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and growth hormone receptor inhibitor treatment.In general, the volunteers treated with placebo have little or noresponse to treatment, whereas the volunteers treated with the growthhormone receptor inhibitor show positive trends in their disease stateor condition index at the conclusion of the study.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RRA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of Growth HormoneReceptor mRNA Levels

Quantitation of growth hormone receptor mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 100 oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human growth hormone receptor were designed tohybridize to a human growth hormone receptor sequence, using publishedsequence information (GenBank accession number NM_000163.1, incorporatedherein as SEQ ID NO:4). For human growth hormone receptor the PCRprimers were:

forward primer: GATGTCCCAATGTGACATGCA (SEQ ID NO: 5)reverse primer: AAGTAGGCATTGTCCATAAGGAAGTT (SEQ ID NO: 6) and the PCRprobe was: FAM-CCGGAAATGGTCTCACTCTGCCAAGA-TAMRA (SEQ ID NO: 7) where FAMis the fluorescent dye and TAMRA is the quencher dye. For human GAPDHthe PCR primers were:forward primer: GAAGGTGAAGGTCGGAGT (SEQ ID NO:8)reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probewas: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE isthe fluorescent reporter dye and TAMRA is the quencher dye.

Probes and primers to mouse growth hormone receptor were designed tohybridize to a mouse growth hormone receptor sequence, using publishedsequence information (GenBank accession number NM_010284.1, incorporatedherein as SEQ ID NO:11). For mouse growth hormone receptor the PCRprimers were:

forward primer: TTGACGAAATAGTGCAACCTGATC (SEQ ID NO:12)reverse primer: CGAATCCCGGTCAAACTAATG (SEQ ID NO: 13) and the PCR probewas: FAM-CATTGGCCTCAACTGGACTTTACTAA-TAMRA (SEQ ID NO: 14) where FAM isthe fluorescent reporter dye and TAMRA is the quencher dye. For mouseGAPDH the PCR primers were:forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:15)reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO:16) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Growth Hormone Receptor mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMARESCO, Inc. Solon, Ohio).RNA was transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human growth hormone receptor, a human growth hormone receptorspecific probe was prepared by PCR using the forward primerGATGTCCCAATGTGACATGCA (SEQ ID NO: 5) and the reverse primerAAGTAGGCATTGTCCATAAGGAAGTT (SEQ ID NO: 6). To normalize for variationsin loading and transfer efficiency membranes were stripped and probedfor human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.).

To detect mouse growth hormone receptor, a mouse growth hormone receptorspecific probe was prepared by PCR using the forward primerTTGACGAAATAGTGCAACCTGATC (SEQ ID NO: 12) and the reverse primerCGAATCCCGGTCAAACTAATG (SEQ ID NO: 13). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Growth Hormone ReceptorExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the human growthhormone receptor RNA, using published sequences (GenBank accessionnumber NM_000163.1, incorporated herein as SEQ ID NO: 4, and thecomplement of positions 468085 to 502183 of the sequence with GenBankaccession number NT_006702.8, incorporated herein as SEQ ID NO: 18). Thecompounds are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humangrowth hormone receptor mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which MCF7 cells were treated with the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”.

TABLE 1 Inhibition of human growth hormone receptor mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS #REGION NO SITE SEQUENCE INHIB ID NO NO 227452 Coding 4 332tcagggcattctttccattc 79 19 1 227453 Coding 4 337 cataatcagggcattctttc 5220 1 227464 Coding 4 947 cctttaatctttggaactgg 58 21 1 227468 Coding 41079 tcatcaatatctagctcaat 62 22 1 227469 Coding 4 1124cttagaagtctgtctgtgtc 63 23 1 227475 Coding 4 1514 cctgctggtgtaatgtcgct68 24 1 227480 Coding 4 1724 atgtaaatgtcctcttggtt 66 25 1 227481 Coding4 1729 tggtgatgtaaatgtcctct 45 26 1 227482 Coding 4 1734ttctgtggtgatgtaaatgt 53 27 1 227483 Coding 4 1739 aggctttctgtggtgatgta75 28 1 227484 Coding 4 1744 tggtaaggctttctgtggtg 63 29 1 227488 Coding4 1922 agttggtctgtgctcacata 86 30 1 227489 Coding 4 1927tgttcagttggtctgtgctc 75 31 1 227490 Coding 4 1936 gcatgattttgttcagttgg67 32 1 227499 3′UTR 4 2656 tataaaagggctttgtaaaa 14 33 1 227500 3′UTR 44043 catagcagcaaagtagcaga 69 34 1 227501 3′UTR 4 4183gctatttttggctatagaaa 64 35 1 227502 3′UTR 4 4197 gattgaggtatttagctatt 5636 1 272302 Start 4 31 gatccatacctgtaggacct 60 37 1 Codon 272303 Start 436 ccagagatccatacctgtag 55 38 1 Codon 272304 Coding 4 115tgctaaggatagctgctgtg 48 39 1 272305 Coding 4 160 ttgtctttaggcctggatta 6840 1 272306 Coding 4 170 ttagaagaatttgtctttag 13 41 1 272307 Coding 4185 gtgaatttaggctccttaga 55 42 1 272308 Coding 4 274gctgtatgggtcctaggttc 57 43 1 272309 Coding 4 362 taacagctgttttccccagc 8544 1 272310 Coding 4 439 tttcatccactgtaccacca 76 45 1 272311 Coding 4468 ttgcactatttcatcaacag 47 46 1 272312 Coding 4 480gggtggatctggttgcacta 57 47 1 272313 Coding 4 564 attgcgtggtgcttcccatc 7748 1 272314 Coding 4 652 tagggtccatcattttccat 56 49 1 272315 Coding 4684 caatgagtacactggaactg 53 50 1 272316 Coding 4 752aactcgccataatttccaga 64 51 1 272317 Coding 4 857 agcccaaatattccaaagat 6552 1 272318 Coding 4 913 tcagcattttaatcctttgc 55 53 1 272319 Coding 4979 attttccttccttgaggaga 67 54 1 272320 Coding 4 1000agattgtgttcacctcctct 70 55 1 272321 Coding 4 1053 aacccaagagtcatcactgt64 56 1 272322 Coding 4 1084 ctggctcatcaatatctagc 84 57 1 272323 Coding4 1110 tgtgtctgattcctcagtct 67 58 1 272324 Coding 4 1236tatgtcattggcattgaaat 53 59 1 272325 Coding 4 1302 aaggcataagagatctgctt66 60 1 272326 Coding 4 1420 actcagctccttcagtagga 77 61 1 272327 Coding4 1560 ggacatccctgccttattct 60 62 1 272328 Coding 4 1623ggcattgtccataaggaagt 85 63 1 272329 Coding 4 1651 actttttggcatctgcctca63 64 1 272330 Coding 4 1656 gatgcactttttggcatctg 47 65 1 272331 Coding4 1861 cagtcgcattgagtatgagg 67 66 1 272332 Coding 4 1884ctctttgtcaggcaagggca 75 67 1 272333 Coding 4 1913 gtgctcacatagccacatga72 68 1 272334 Stop 4 1949 aagaaaggctaaggcatgat 61 69 1 Codon 2723353′UTR 4 1973 aaatacgtagctcttgggaa 47 70 1 272336 3′UTR 4 2196caatcactgctactaaacag 69 71 1 272337 3′UTR 4 2249 aaacatagccattcaatgct 3972 1 272318 3′UT1 4 2337 gtgctatggtttgcattcaa 78 73 1 272339 3′UTR 42454 gttttacatatccaaactat 72 74 1 272340 3′UTR 4 2853catcaaccaagatttggtga 69 75 1 272341 3′UTR 4 2988 gaggctatagatcttatctc 6576 1 272342 31UTR 4 3271 tagtgagaaagaaagtttct 45 77 1 272343 3′UTR 43765 aatgctctcaagaatgatgt 48 78 1 272344 3′UTR 4 3980acactcaattctagcttttc 60 79 1 272345 3′UTR 4 4011 catctattacaaataacatg 2480 1 272346 3′UTR 4 4057 ctcttggagaaaaccatagc 67 81 1 272347 3′UTR 44097 tctacactgatgatacttta 62 82 1 272348 3′UTR 4 4120cacagctttgaattgaatta 57 83 1 272349 3′UTR 4 4133 agtcttccaaacacacagct 6884 1 272350 3′UTR 4 4156 aggctgttgtgaaatagtaa 67 85 1 272351 3′UTR 44170 atagaaatgttgtcaggctg 57 86 1 272352 3′UTR 4 4218ccaaaatgacattctgagac 77 87 1 272353 3′UTR 4 4245 ataatggcttatgtggccac 7288 1 272354 intron 18 2571 agttatgtgaccctgattga 65 89 1 272355 intron:18 6418 ttgagtgttcctaaaatgaa 24 90 1 exon junction 272356 intron 18 8405atggaggctggaggttcaaa 63 91 1 272357 intron: 18 22712tagggtccatctttcaagac 62 92 1 exon junction 272358 intron 18 25543tctccagatagaatctaaac 53 93 1 272359 intron 18 29755 tccaaatattctggtacttt72 94 1 272360 exon: 18 29935 tattagttaccttgaggaga 0 95 1 intronjunction 272361 intron: 18 30267 attttccttcctagaaaata 10 96 1 exonjunction

As shown in Table 1, SEQ ID NOs 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 73, 74, 75, 76, 77, 78, 79, 81, 82, 83, 84, 85, 86,87, 88, 89, 91, 92, 93 and 94 demonstrated at least 45% inhibition ofhuman growth hormone receptor expression in this assay and are thereforepreferred. More preferred are SEQ ID NOs 30, 44 and 57.

ISIS 272322 (SEQ ID NO: 57) is targeted to exon 10, a region whichappears in all growth hormone receptor transcripts. Compounds targetedto exon 10 are therefore preferred embodiments of the invention. Exon 3,reported to be alternatively spliced in the human transcript(s), mayalso be a preferred target region.

The target regions to which the preferred antisense sequences of Table 2are complementary are herein referred to as “preferred target segments”and are therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 3. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 3 is the species in whicheach of the preferred target segments was found.

Example 16 Antisense Inhibition of Mouse Growth Hormone ReceptorExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a second series of antisensecompounds were designed to target different regions of the mouse growthhormone receptor RNA, using published sequences (GenBank accessionnumber NM_010284.1, incorporated herein as SEQ ID NO: 11, a variant ofGenBank accession number AF120480.2 with an alternative splice site fromexon 1B:exon 2, incorporated herein as SEQ ID NO: 97, a variant ofGenBank accession number AF120480.2 with an alternative splice site atfrom exon 1C:exon 2, incorporated herein as SEQ ID NO: 98, a variant ofGenBank accession number AF120480.2 with an alternative splice site fromexon 1D:exon 2, incorporated herein as SEQ ID NO: 99, and a sequencederived from GenBank accession numbers AF120480.2 and AC073753.1,representing a genomic sequence, incorporated herein as SEQ ID NO: 100).The compounds are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mousegrowth hormone receptor mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from threeexperiments in which b.END cells were treated with the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse growth hormone receptor mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS #REGION NO SITE SEQUENCE INHIB ID NO NO 227443 5′UTR 11 5tgcttggcagctcgtgggtt 0 101 1 227444 5′UTR 11 16 atggctgcgcctgcttggca 53102 1 227445 Start 11 221 tacctgagacctcggagttt 69 103 1 Codon 227446Start 11 232 acaaagatccatacctgaga 87 104 1 Codon 227447 Coding 11 300gctggtgtagcctcacttcc 77 105 1 227448 Coding 11 313 tttgccaagagtagctggtg60 106 1 227449 Coding 11 391 acgacacttggtgaatcgag 69 107 1 227450Coding 11 495 tggctttcccttttagcata 71 108 1 227451 Coding 11 520atgagcaattcttgcagctt 49 109 1 227454 Coding 11 590 agttgaagtaacagctgttt69 110 1 227455 Coding 11 620 agtagggtatccaaatggag 43 111 1 227456Coding 11 717 gtccagttgaggccaatggg 97 112 1 227457 Coding 11 812gaattatccatcccttcaga 67 113 1 227458 Coding 11 832 gtactgaatttcatactcca75 114 1 227459 Coding 11 975 ctgaactcgctgtacttttc 60 115 1 227460Coding 11 1041 aactggatatcttcttcaca 43 116 1 227461 Coding 11 1084tgctactccaaatattccaa 75 117 1 227462 Coding 11 1115 gctttgaaaatataactaca31 118 1 227463 Coding 11 1137 atcagcatcttaatcctttg 39 119 1 227465Coding 11 1190 tgagaagatctggatcaatc 51 120 1 227466 Coding 11 1245ttgtagttatcatgaatgcc 50 121 1 227467 Coding 11 1265 catcattgtagaagtcgggt33 122 1 227470 Coding 11 1388 ctccaaggataccagctgat 82 123 1 227471Coding 11 1530 aggcacaagagatcagcttc 52 124 1 227472 Coding 11 1579agagccaagggaagcatcat 42 125 1 227473 Coding 11 1710 aagtcaatgtttgccagtga71 126 1 227474 Coding 11 1730 tgtcgcttacttgggcataa 68 127 1 227476Coding 11 1837 gtaattttcttggcagggcg 41 128 1 227477 Coding 11 1850cactgttcatgctgtaattt 61 129 1 227478 Coding 11 1878 tttttggcatctgactcaca68 130 1 227479 Coding 11 1947 atgtcctcttggttaaagct 59 131 1 227485Coding 11 2044 cgtggtgtagtctgggacag 45 132 1 227486 Coding 11 2054cggtgtgaaccgtggtgtag 39 133 1 227487 Coding 11 2106 tcaggcaaaggcaaagcagt44 134 1 227491 Stop 11 2182 taggaaaggctactgcatga 65 135 1 Codon 2274923′UTR 11 2239 taaaacatagttttggttta 7 136 1 227493 3′UTR 11 2253tcccaacacagatttaaaac 51 137 1 227494 3′UTR 11 2517 caaaagccacctgattgttt56 138 1 227495 3′UTR 11 2527 tcctgaactgcaaaagccac 47 139 1 227496 3′UTR11 2537 gcattcaatttcctgaactg 51 140 1 227497 3′UTR 11 2637taaatgttttgcatatccaa 77 141 1 227498 3′UTR 11 2824 ttgtaaaaatctaacttgtt49 142 1 227503 exon: 97 197 tacctgagaccccagttcat 24 143 1 exon junction227504 exon: 98 23 tacctgagaccccgcgcagc 34 144 1 exon junction 227505exon: 99 61 tacctgagacccacaagcgg 39 145 1 exon junction 227506 exon: 1004352 cctccagtacctcggagttt 69 146 1 intron junction 227507 intron: 1004865 gtccttgctccaggttagca 89 147 1 exon junction 227508 exon: 100 5071ttccactcaccccagttcat 51 148 1 intron junction 227509 intron: 100 5153gcagttctatcagaactttg 82 149 1 exon junction 227510 intron 100 5196ctccagacgtgacccgactc 64 150 1 227511 exon: 100 5264 ccacgcacccacaagcggat71 151 1 intron junction 227512 intron 100 6350 taacctatggtgactatgtc 36152 1 227513 intron: 100 7123 tacctgagacctgcaagaca 40 153 1 exonjunction 227514 intron 100 9753 atgctcacgtcagctattgg 43 154 1 227515exon: 100 13932 aaattcttacttgtccccag 37 155 1 intron junction 227516intron: 100 17200 ttggctttccctggaggttc 57 156 1 exon junction 227517exon: 100 17224 cttcactaaccttgcagctt 63 157 1 intron junction 227518exon: 100 24259 cacggcttacctatttcgtc 6 158 1 intron junction 227519exon: 100 37843 tcacacctacctttgctgct 44 159 1 intron junction 227520intron: 100 40862 catcttaatccttggaaaca 42 160 1 exon junction

As shown in Table 2, SEQ ID NOs 102, 103, 104, 105, 106, 107, 108, 110,112, 113, 114, 115, 117, 120, 121, 123, 124, 126, 127, 129, 130, 131,135, 137, 138, 140, 141, 146, 147, 148, 149, 150, 151, 156 and 157demonstrated at least 50% inhibition of mouse growth hormone receptorexpression in this experiment and are therefore preferred. Morepreferred are SEQ ID NOs 104, 147, and 149.

ISIS 227446, 227507 and 227509 (SEQ ID NO: 104, 147 and 149) weresubjected to dose-response studies. All three compounds showed good doseresponses with IC50s of approximately 25 nM, 12.5 nM and 12.5 nM,respectively. The target regions to which the preferred antisensesequences of Table 2 are complementary are herein referred to as“preferred target segments” and are therefore preferred for targeting bycompounds of the present invention. These preferred target segments areshown in Table 3. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 2. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 3 is thespecies in which each of the preferred target segments was found.

TABLE 3 Sequence and position of preferred target segments identifiedin growth hormone receptor. TARGET SITE SEQ ID TARGET REV COMP SEQ ID IDNO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 144070 4 332gaatggaaagaatgccctga 19 H. sapiens 161 144071 4 337 gaaagaatgccctgattatg20 H. sapiens 162 144082 4 947 ccagttccaaagattaaagg 21 H. sapiens 163144086 4 1079 attgagctagatattgatga 22 H. sapiens 164 144087 4 1124gacacagacagacttctaag 23 H. sapiens 165 144093 4 1514agcgacattacaccagcagg 24 H. sapiens 166 144098 4 1724aaccaagaggacatttacat 25 H. sapiens 167 144099 4 1729agaggacatttacatcacca 26 H. sapiens 168 144100 4 1734acatttacatcaccacagaa 27 H. sapiens 169 144101 4 1739tacatcaccacagaaagcct 28 H. sapiens 170 144102 4 1744caccacagaaagccttacca 29 H. sapiens 171 144106 4 1922tatgtgagcacagaccaact 30 H. sapiens 172 144107 4 1927gagcacagaccaactgaaca 31 H. sapiens 173 144108 4 1936ccaactgaacaaaatcatgc 32 H. sapiens 174 144118 4 4043tctgctactttgctgctatg 34 H. sapiens 175 144119 4 4183tttctatagccaaaaatagc 35 H. sapiens 176 144120 4 4197aatagctaaatacctcaatc 36 H. sapiens 177 188518 4 31 aggtcctacaggtatggatc37 H. sapiens 178 188519 4 36 ctacaggtatggatctctgg 38 H. sapiens 179188520 4 115 cacagcagctatcettagca 39 H. sapiens 180 188521 4 160taatccaggcctaaagacaa 40 H. sapiens 181 188523 4 185 tctaaggagcctaaattcac42 H. sapiens 182 188524 4 274 gaacctaggacccatacagc 43 H. sapiens 183188525 4 362 gctggggaaaacagctgtta 44 H. sapiens 184 188526 4 439tggtggtacagtggatgaaa 45 H. sapiens 185 188527 4 468 ctgttgatgaaatagtgcaa46 H. sapiens 186 188528 4 480 tagtgcaaccagatccaccc 47 H. sapiens 187188529 4 564 gatgggaagcaccacgcaat 48 H. sapiens 188 188530 4 652atggaaaatgatggacccta 49 H. sapiens 189 188531 4 684 cagttccagtgtactcattg50 H. sapiens 190 188532 4 752 tctggaaattatggcgagtt 51 H. sapiens 191188533 4 857 atctttggaatatttgggct 52 H. sapiens 192 188534 4 913gcaaaggattaaaatgctga 53 H. sapiens 193 188535 4 979 tctcctcaaggaaggaaaat54 H. sapiens 194 188536 4 1000 agaggaggtgaacacaatct 55 H. sapiens 195188537 4 1053 acagtgatgactcttgggtt 56 H. sapiens 196 188538 4 1084gctagatattgatgagccag 57 H. sapiens 197 188539 4 1110agactgaggaatcagacaca 58 H. sapiens 198 188540 4 1236atttcaatgccaatgacata 59 H. sapiens 199 188541 4 1302aagcagatctettatgectt 60 H. sapiens 200 188542 4 1420tcctactgaaggagctgagt 61 H. sapiens 201 188543 4 1560agaataaggcagggatgtcc 62 H. sapiens 202 188544 4 1623acttccttatggacaatgcc 63 H. sapiens 203 188545 4 1651tgaggcagatgccaaaaagt 64 H. sapiens 204 188546 4 1656cagatgccaaaaagtgcatc 65 H. sapiens 205 188547 4 1861cctcatactOaatgcgactg 66 H. sapiens 206 188548 4 1884tgcccttgcCtgacaaagag 67 H. sapiens 207 188549 4 1913tcatgtggctatgtgagcac 68 H. sapiens 208 188550 4 1949atcatgccttagcctttctt 69 H. sapiens 209 188551 4 1973ttcccaagagctacgtattt 70 H. sapiens 210 188552 4 2196ctgtttagtagcagtgattg 71 H. sapiens 211 188554 4 2337ttgaatgcaaaccatagcac 73 H. sapiens 212 188555 4 2454atagtttggatatgtaaaac 74 H. sapiens 213 188556 4 2853tcaccaaatcttggttgatg 75 H. sapiens 214 188557 4 2988gagataagatctatagcctc 76 H. sapiens 215 188558 4 3271agaaactttctttctcacta 77 H. sapiens 216 188559 4 3765acatcattcttgagagcatt 78 H. sapiens 217 188560 4 3980gaaaagctagaattgagtgt 79 H. sapiens 218 188562 4 4057gctatggttttctccaagag 81 H. sapiens 219 188563 4 4097taaagtatcatcagtgtaga 82 H. sapiens 220 188564 4 4120taattcaattcaaagctgtg 83 H. sapiens 221 188565 4 4133agctgtgtgtttggaagact 84 H. sapiens 222 188566 4 4156ttactatttcacaacagcct 85 H. sapiens 223 188567 4 4170cagcctgacaacatttctat 86 H. sapiens 224 188568 4 4218gtctcagaatgtcattttgg 87 H. sapiens 225 188569 4 4245gtggccacataagccattat 88 H. sapiens 226 188570 18 2571tcaatcagggtcacataact 89 H. sapiens 227 188572 18 8405tttgaacctccagcctccat 91 H. sapiens 228 188573 18 22712gtcttgaaagatggacccta 92 H. sapiens 229 188574 18 25543gtttagattctatctggaga 93 H. sapiens 230 188575 18 29755aaagtaccagaatatttgga 94 H. sapiens 231 144062 11 16 tgccaagcaggcgcagccat102 M. musculus 232 144063 11 221 aaactccgaggtctcaggta 103 M. musculus233 144064 11 232 tctcaggtatggatctttgt 104 M. musculus 234 144065 11 300ggaagtgaggctacaccagc 105 M. musculus 235 144066 11 313caccagctactcttggcaaa 106 M. musculus 236 144067 11 391ctcgattcaccaagtgtcgt 107 M. musculus 237 144068 11 495tatgctaaaagggaaagcca 108 M. musculus 238 144072 11 590aaacagctgttacttcaact 110 M. musculus 239 144074 11 717cccattggcctcaactggac 112 M. musculus 240 144075 11 812tctgaagggatggataattc 113 M. musculus 241 144076 11 832tggagtatgaaattcagtac 114 M. musculus 242 144077 11 975gaaaagtacagcgagttcag 115 M. musculus 243 144079 11 1084ttggaatatttggagtagca 117 M. musculus 244 144083 11 1190gattgatccagatcttctca 120 M. musculus 245 144084 11 1245ggcattcatgataactacaa 121 M. musculus 246 144088 11 1388atcagctggtatccttggag 123 M. musculus 247 144089 11 1530gaagctgatctcttgtgcct 124 M. musculus 248 144091 11 1710tcactggcaaacattgactt 126 M. musculus 249 144092 11 1730ttatgcccaagtaagcgaca 127 M. musculus 250 144095 11 1850aaattacagcatgaacagtg 129 M. musculus 251 144096 11 1878tgtgagtcagatgccaaaaa 130 M. musculus 252 144097 11 1947agctttaaccaagaggacat 131 M. musculus 253 144109 11 2182tcatgcagtagcctttccta 135 M. musculus 254 144111 11 2253gttttaaatctgtgttggga 137 M. musculus 255 144112 11 2517aaacaatcaggtggcttttg 138 M. musculus 256 144114 11 2537cagttcaggaaattgaatgc 140 M. musculus 257 144115 11 2637ttggatatgcaaaacattta 141 M. musculus 258 144124 100 4352aaactccgaggtactggagg 146 M. musculus 259 144125 100 4865tgctaacctggagcaaggac 147 M. musculus 260 144126 100 5071atgaactggggtgagtggaa 148 M. musculus 261 144127 100 5153caaagttctgatagaactgc 149 M. musculus 262 144128 100 5196gagtcgggtcacgtctggag 150 M. musculus 263 144129 100 5264atccgcttgtgggtgcgtgg 151 M. musculus 264 144134 100 17200gaacctccagggaaagccaa 156 M. musculus 265 144135 100 17224aagctgcaaggttagtgaag 157 M. musculus 266

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of growth hormonereceptor.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 17 Western Blot Analysis of Growth Hormone Receptor ProteinLevels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to growth hormonereceptor is used, with a radiolabeled or fluorescently labeled secondaryantibody directed against the primary antibody species. Bands arevisualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

Example 18 Reduction of Serum IGF-I in Animals after Treatment withAntisense to Growth Hormone Receptor-1 Week Pilot Study

Forty male Balb/C(a) mice weighing 9 to log were placed into cages, 4animals per cage, and allowed to assimilate into their environment withnew littermates ˜1 week prior (Day −7) to the commencement of 1 weekstudy. Mice of this age would be at their maximum growth rate. Theirbody weights were measured and recorded every second day during thisperiod. When mice weighed 11 g (day −2), a blood sample was collectedunder anesthesia as described below, and a serum IGF-I assay wasperformed to determine pre-treatment values and to aid in the assigningof mice to treatment groups in order to reduce animal variability. Toobtain the blood sample, the animals were anaesthetized withpentobarbital (50 mg/kg i.p.) and non-fasting blood samples collectedexactly 5 minutes later from the retrobulbar plexus through heparinizedcapillary tubes under light ether anesthesia. The 40 animals were placedinto five groups with each group having a similar weight average andsimilar IGF-I average concentration for the trial.

Animals (n=8/group) were designated to the following five treatmentgroups:

Control—saline (once every 2 days)

ASO (Antisense to growth hormone receptor)—ISIS 227446 (SEQ ID NO: 104)(3 and 30 mg/kg once every 2 days)

Mismatch (negative control oligonucleotide)—ISIS 261303 (SEQ ID NO: 267,8-base mismatch to ISIS 227446) (30 mg/kg once every 2 days)

Octreotide—(25 μg/kg/twice per day)

Saline, antisense, mismatch control and octreotide samples wereprepared, and coded for blinding. Animals were given a subcutaneous doseof saline every second day, and mismatch control or antisense withadministration on days 0, 2, 4, 6. Animals were given twice daily dosesof 25 μg octreotide. Animals were housed 4 per cage, for the duration ofone week. They had access to a pre-determined quantity of standard mousefood and water at all times throughout the experiment. They were housedin a quiet, temperature- and humidity-maintained environment for theentirety of the study. At day 0 and before treatment on each day orsecond day, the animals had their body weight and food intake measured,enabling the correct dose of agent to be administered. The animals weremonitored closely for any changes in fur, skin, eye, locomotion or otherchanges in behavior. No problems were observed. Every second day fromday −7 to day 7 body weight and food intake were measured.

On day 7, one day after the last dose of antisense, and/or after thelast octreotide dose, the animals were anaesthetized with pentobarbital(50 mg/kg i.p.) and non-fasting blood samples collected exactly 5minutes later from the retrobulbar plexus through heparinized capillarytubes under light ether anesthesia (as on day −7 and 0).

At day −2 and day 7, serum IGF-I measurement was done byradioimmunoassay. The results are shown in Table 4. Serum IGF-I level isthe most widely used measure of growth hormone biological activity inhuman therapy. It is used to measure the efficacy of growth hormoneantagonist treatments like Trovert, which block cells' responsiveness toexcess growth hormone, and dopamine agonists and octreotide somatostatinantagonist drugs that block growth hormone secretion from the pituitary.

TABLE 4 Effect of antisense inhibitor of growth hormone receptor onserum insulin-like growth factor-I levels IGF-I (ng/ml) IGF-I (ng/ml) %IGF-I Day −2 Day 7 reduction* Saline Control 217.09 ± 42.61 102.64 ±31.64 0   Octreotide 199.72 ± 44.47 114.34 ± 41.36 — ASO 3 mg/kg 216.23± 78.14 129.63 ± 33.76 — ASO 30 mg/kg 181.84 ± 71.32  56.95 ± 10.3444.51 Mismatch 184.87 ± 55.6   81.1 ± 19.16 20.98 30 mg/kg *Percentreduction in serum IGF-I at day 7 compared to saline control at day 7.

As shown in Table 4, the growth hormone receptor antisense compound,ISIS 227446 (SEQ ID NO: 104, dosed subcutaneously at 30 mg/kg everysecond day for one week, produced a statistically significant andspecific reduction of serum IGF-I to 55% of the control (saline) group.By t-test the antisense 30 mg/kg was significantly different from thesaline control (p<0.005) and the mismatch control (p<0.01). The mismatchcontrol was not statistically different from the saline control(p>0.05). There was no effect at 3 mg/kg. The 45% reduction in serumIGF-I levels in our study using 30 mg/kg antisense every second day iscomparable to that achieved using 10 mg/kg daily Trovert (Van Neck etal., J. Endocrinol., 2000, 167, 295-303).

The negative control 8-nucleotide mismatch oligonucleotide ISIS 261303(SEQ ID NO: 267), reduced serum IGF-I by 21% compared to the controlsaline group, however, this reduction was not statistically significant(with p>0.05). Octreotide, 2 doses per day at 25 μg each had no effecton serum IGF-I levels at day 7. The non-effect obtained with octreotideis consistent with data reported by Groenbaek et al. (J. Endocrinol.,2002, 172, 637-643) using this dose and twice this dose at day 7 indiabetic animals. In diabetic animals two 50 μg doses of octreotide perday for two weeks are required to reduce sIGF-I levels.

Thus an antisense inhibitor of growth hormone receptor has now beendemonstrated to specifically reduce serum insulin-like growth factor-Ilevels by 45% compared to control. Reduction of serum insulin-likegrowth factor-I by similar levels using octreotide or Trovert, areclinically relevant in the treatment of diseases including acromegaly,gigantism, age-related macular degeneration, diabetic retinopathy,diabetic nephropathy, diabetes, and growth hormone and IGF-I-dependenttumors as outlined supra. Thus antisense therapy is believed to betherapeutically useful for treatment of conditions associated with thegrowth hormone/insulin-like growth factor-I axis.

The serum remaining following the insulin-like growth factor-1 assay wasisolated and stored at −80° C. The whole liver was removed rapidly forweighing and snap-frozen in labelled aluminum parcels by submersion inliquid-nitrogen. Kidney and spleen were also snap frozen in liquidnitrogen and stored at −80° C. in the freezer. The carcass was weighedand then placed into a sealable plastic bag, snap-frozen on dry ice andkept at −80° C.

The decline in serum insulin-like growth factor-I with 30 mg/kg ofantisense was not sufficient to influence body weight or organ weightsover this period. This confirms published results of others. Van Neck etal., J Endocrinol., 2000, 167, 295-303. Looking at the study overall,body length increase during the study was in the range 7.5-10%. Taillength increases were in proportion to overall length increases. Foodintake did not vary significantly between treatment groups. Growth (bodylength and weight) were unaffected by any treatment. Weight was measuredin two ways: weight trend (live animal), and final carcass weight.Absolute liver weights were unchanged except for a slight increase inliver weight (g/total body weight) for the octreotide group. Weights ofother organs were unaffected. These observations were similar to thosereported by van Neck et al. with Trovert except that liver weight wasunaffected by Trovert, as also observed with growth hormone receptorantisense.

Growth hormone receptor mRNA levels in tissue samples from our currentstudy are assayed from liver and kidney to test for an RNase H-basedantisense mechanism of action. Growth hormone receptor protein levels byWestern or binding assays in tissue samples from our current study areassayed from liver and/or kidney to test for additional and/oralternative antisense mechanisms of action. Liver contributes to 75% ofserum insulin-like growth factor-I levels as shown in growth hormonereceptor knockout animals of Sjogren et al., Proc. Natl. Acad. Sci. USA,1999, 96, 7088-7092. Sample analysis of the liver and kidneyinsulin-like growth factor-I by Western and Northern blot total RNAanalysis or quantitative PCR is also done as would be understood bythose skilled in the art.

Example 19 Reduction of Growth Hormone Receptor Activity in Animalsafter Treatment with Antisense to Growth Hormone Receptor

Specific binding assays were carried out with liver tissue usingiodinated human growth hormone [¹²⁵I] hGH.

Microsomal membrane preparations were obtained as follows. 400 mg oftissue powder was homogenized in cold homogenizing buffer (50 mMTris/HCl, 250 mM sucrose, pH 7.4). This was centrifuged at 2000 rpm for10 min at 3° C. and the supernatant was saved. This was centrifugedagain at 15,000 rpm for 20 min. Pellets were resuspended in 0.5 ml ofRRA buffer with inhibitor (50 mM Tris, 20 mM MgCl₂, pH 7.4). Microsomalpreparation samples were stored at −80° C. until the specific bindingassay.

The [¹²⁵I] hGH specific binding assay was done as follows. Four glasstubes were set up for each sample, two for (−), two for (+). Differentsample and solutions were added in each tube as follows (i) 0.2 ml RRAbuffer (50 mM Tris, 20 mM MgCl₂, 0.1% BSA, pH 7.4); (ii) 0.1 ml membrane(½ or ¼ dilution); (iii) 0.1 ml bGH (10 μg/ml) for the (+) tube or 0.1ml RRA buffer for the (−) tube; and (iv) 0.1 ml [¹²⁵I]-hGH tracer.

Samples were incubated at 4° C. with shaking overnight. The reaction wasstopped with 2.5 ml of cold RRA, and the sample centrifuged at 2800 rpmfor 25 min at 4° C. Supernatant was aspirated and pellets counted usingthe γ-counter. The specific binding capacity was calculated as:CPM(−)-CPM(+). Protein content of the microsomal samples was determinedby the BCA protein assay.

TABLE 5 Effect of antisense inhibitor on growth hormone receptor growthhormone binding activity Specific binding/mg Specific binding/mg protein(cpm) protein (cpm) ½ dilution ¼ dilution Saline Control 5647 ± 746 9071± 2371 ASO 30 mg/kg 4205 ± 534 (26% 5546 ± 789 (39% reduction comparedreduction compared to saline) to saline) Mismatch 30 mg/kg 7090 ± 18778431 ± 2663

As shown in Table 5, the growth hormone receptor antisense compound,ISIS 227446 (SEQ ID NO: 104, dosed subcutaneously at 30 mg/kg everysecond day for one week, produced a statistically significant (p<0.05)and specific reduction of growth hormone receptor levels (measured bygrowth hormone binding activity) to 61% of control (saline) group. Thenegative control 8-nucleotide mismatch oligonucleotide ISIS 261303 (SEQID NO: 267) had no effect compared to the control saline group. Theantisense inhibitor of growth hormone receptor produced a statisticallysignificant (p<0.01) and specific reduction of growth hormone receptorlevels to 59% of the control (mismatch) group in the ½ dilutionexperiment.

The specific reduction of growth hormone receptor levels wassignificantly (by t-test) different from both the saline control and themismatch control at both dilutions (p<0.05).

These growth hormone receptor level measurements following antisensetreatment are consistent with the 45% reduction in serum insulin-likegrowth factor-I levels in our study using 30 mg/kg antisense everysecond day relative to control (saline).

Example 20 Reduction of Growth Hormone Receptor mRNA Levels and SerumIGF-I in Animals after Treatment with Antisense to Growth HormoneReceptor—Additional 1 Week Study

Male Balb/C(a) mice were prepared and grouped for analysis as in Example18 above.

Animals (n=10/group) were designated to the following treatment groups:

Control—saline (once every 2 days)

ASO (Antisense to growth hormone receptor)—ISIS 227446 (SEQ ID NO: 104)(30 and 50 mg/kg once every 2 days)

Unrelated negative control oligonucleotide—ISIS 260120(TTACCGTATGGTTCCTCACT; SEQ ID NO: 268, (50 mg/kg once every 2 days)

Animals were treated and serum IGF-I levels were measured as in Example18 above. Briefly, for the one-week study, mice were given asubcutaneous dose of saline every second day, and mismatch control orantisense with administration on days 0, 2, 4, 6. On day 7, the animalswere anaesthetized with pentobarbital and non-fasting blood samplescollected exactly 5 minutes later from the retrobulbar plexus throughheparinized capillary tubes under light ether anesthesia. Serum IGF-Imeasurement was done by radioimmunoassay at day 7.

In the one-week study, the growth hormone receptor antisense inhibitorISIS 227446 reduced serum IGF-I by 33% at the 50 mg/kg dose, relative tosaline control (p<0.001), and by 20% relative to the unrelated control(p<0.068). The unrelated control at the 50 mg/kg dose reduced serumIGF-I by 17% compared to saline (p>0.05).

Growth hormone receptor mRNA levels in liver tissue samples from treatedand untreated mice in this one-week study were assayed. The growthhormone receptor antisense inhibitor ISIS 227446 reduced growth hormonereceptor mRNA levels in liver after the one-week study by 72% at the 50mg/kg dose, relative to saline control (p<0.0001). The 30 mg/kg dose ofISIS 227446 yielded a 50% decrease in growth hormone receptor mRNA(p<0.0001). The unrelated control oligonucleotide ISIS 260120, at 50mg/kg, reduced growth hormone receptor mRNA levels by approximately 15%(p>0.05).

Example 21 Reduction of Growth Hormone Receptor mRNA Levels and SerumIGF-I in Animals after Treatment with Antisense to Growth HormoneReceptor—2 Week Study

A two-week study was done in similar fashion to the one-week study inExample 18, this time using ISIS 227446 at doses of 3, 5, 10, 20 and 30mg/kg. The mismatch control was given at the same doses. Mice weretreated with antisense compound or saline every other day for 14 days.

Table 5 shows the serum IGF-I levels in mice treated for 14 days.P-values were determined by t-test.

TABLE 5 Two week mouse study-serum IGF-I levels after treatment withantisense inhibitor of growth hormone receptor Dose of ISIS Day 14 %decrease 227446 serum IGF- relative to 3 mg/kg (mg/kg) I ng/ml ISIS227446 p-value 30 126 41 0.0002 20 122 43 0.0002 10 130 39 0.0002 5 1949 0.3261 3 214 0 —

The reduction in serum IGF-I at 14 days was dependent on dose with39-43% decrease in levels achieved at >10 mg/kg compared to 3 mg/kg. The3 mg/kg dose of ISIS 227446 had no effect on serum IGF-I levels and wasequivalent to saline (untreated) control (shown in separate experiment).

Mismatch controls gave lesser reductions in serum′IGF-I levels. Theseresults are shown in Table 6. The effect at 30 mg/kg observed with themismatch oligonucleotide at 2 weeks was not observed with an unrelatednegative control oligonucleotide (ISIS 260120; SEQ ID NO: 268).

TABLE 6 Two week mouse study-serum IGF-I levels after treatment withmismatch control ISIS 261303 Dose of ISIS Day 14 % decrease 261303 serumIGF- relative to 3 mg/kg (mg/kg) I ng/ml ISIS 261303 p-value 30 130 290.0094 20 164 11 0.2496 10 174 5 0.6160 5 186 0 0.9359 3 184 0 —

Growth hormone receptor mRNA levels in liver tissue samples from treatedand untreated mice in this two-week study were assayed. The growthhormone receptor antisense inhibitor ISIS 227446 reduced growth hormonereceptor mRNA levels in liver after the two-week study by 50% at the 20mg/kg dose relative to saline control (p<0.001). The 30 mg/kg dose ofISIS 227446 yielded a 53% decrease in growth hormone receptor mRNA(p<0.0001). The mismatch control oligonucleotide ISIS 261303 (SEQ ID NO:267), at 30 mg/kg, reduced growth hormone receptor mRNA levels byapproximately 3%.

Example 22 Effect of Antisense Inhibition of Growth Hormone Receptor onRetinopathy

Retinopathy of prematurity is a neovascularization disorder that canlead to blindness in very low birth weight infants. The retinopathy(abnormal blood vessel formation) is initiated by relatively high oxygenlevels such as are found in infant incubators, for example. A mousemodel of retinopathy (abnormal blood vessel formation in the retina) isused to study the effects of drugs on the extent of neovascularization.

Seven-day-old mice are placed in an infant incubator with their nursingmother in 75% oxygen from postnatal day 7 to day 12 to produceoxygen-induced retinopathy as described in the literature. Smith et al.,1994, Invest Ophthalmol Vis Sci 35,101-111; Robinson et al., Proc NatlAcad Sci USA., 1996, May 14; 93, 4851-6. Oxygen concentration ismeasured at least daily while the animals are in oxygen. On postnatalday 12, the animals are returned to room air. Animals are sacrificed onpostnatal day 17 when maximal neovascularization is observed.

Mice are dosed with antisense oligonucleotide at postnatal days 12, 13,14, 15, and 16 or days 7, 8, 9, 11, 13, 15 and 17. Oligonucleotide isadministered intraperitoneally at concentrations of 5, 10, 20 and 30mg/kg. The mismatch control ISIS 261303 and/or the unrelated negativeantisense control ISIS 260120 are also given.

Example 23 Additional Models

Studies using antisense inhibitors of growth hormone receptor are alsodone in the following pathology animal models and in humans as would beunderstood by those skilled in the art: diabetic nephropathy type I andtype II models, cancer models, arthritis models and chemotherapy induceddiarrhea models.

1-23. (canceled)
 24. A method of reducing the serum level of growthhormone binding protein in a human subject comprising: identifying ahuman subject in need of a reduction in said subject's serum level ofgrowth hormone binding protein; and administering to said subject inneed thereof an oligonucleotide conjugate wherein the oligonucleotide isa modified oligonucleotide 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases inlength, wherein said modified oligonucleotide is fully complementaryover the entirety of said modified oligonucleotide to a human growthhormone receptor RNA, wherein said modified oligonucleotide comprises atleast one modified internucleoside linkage, or at least one modifiedsugar moiety, or at least one modified nucleobase, and wherein saidsubject's serum level of growth hormone binding protein is reduced.25-45. (canceled)
 46. The method of claim 24, wherein theoligonucleotide conjugate has enhanced cellular distribution comparedwith said modified oligonucleotide.
 47. The method of claim 24, whereinthe oligonucleotide conjugate has enhanced cellular distribution to theliver, fat and/or kidney compared with said modified oligonucleotide.48. The method of claim 24, wherein the oligonucleotide conjugate hasenhanced cellular distribution to the liver.
 49. The method of claim 48,wherein the oligonucleotide has enhanced cellular distribution tohepatocytes of the liver.
 50. The method of claim 24, wherein theoligonucleotide conjugate has enhanced cellular uptake compared withsaid modified oligonucleotide.
 51. The method of claim 24, wherein theoligonucleotide conjugate is an oligonucleotide-cholesterol conjugate.52. The method of claim 24, wherein said modified oligonucleotide is anantisense oligonucleotide, a DNA oligonucleotide, a RNA oligonucleotide,a chimeric oligonucleotide, or a short interfering RNA molecule.
 53. Themethod of claim 52, wherein said modified oligonucleotide is a duplexedoligonucleotide or a single stranded oligonucleotide.
 54. The method ofclaim 24, wherein said modified oligonucleotide comprises at least onemodification selected from the group consisting of a2′-O-(2-methoxyethyl) sugar moiety, a 2′-O-methoxy sugar moiety, aphosphorothioate internucleoside linkage, and a 5-methylcytosine. 55.The method of claim 54, wherein said modified oligonucleotide comprises:a region of deoxynucleotides flanked on the 5′ and the 3′ ends of saidregion with a 5′ region and a 3′ region, each of which 5′ region and 3′region comprises at least one 2′-O-(2-methoxyethyl) or 2′-O-methoxynucleotide.
 56. The method of claim 55, wherein said modifiedoligonucleotide consists of 20 linked nucleosides.